JP6356227B2 - Inhibitors of metallo β-lactamase - Google Patents

Description

RELATED APPLICATION This application claims the benefit of priority of pending US Provisional Application No. 61 / 820,277, filed May 7, 2013, the contents of which are hereby incorporated by reference. Incorporated throughout.

  This application relates to combination treatments for bacterial infections. For example, the present application provides one or more antibiotics for treating a disease, disorder, or condition resulting from an infection of a bacterial expressing a metallo β-lactamase or a bacterial infection expressing a metallo β-lactamase and the present specification Relates to the use of one or more compounds of the formula I as defined in the text.

Beta-lactam antibiotics (beta-lactam) (penicillin, cephalosporin, carbapenem, and monobactam) are the most important and frequently used antibiotics in medicine, especially in the treatment of severe Gram-negative bacterial infections One of the classes of agents. From the clinical introduction of penicillin and cephalosporin over the past 60 years, there have been ongoing clinical problems due to the emergence of β-lactamase, an enzyme that hydrolyzes the β-lactam ring involved in the cell killing activity of these compounds. Happening ¹ Antibiotic resistance has enhanced medical and chemical efforts to broaden the antimicrobial spectrum while protecting the core β-lactam backbone from hydrolysis catalyzed by β-lactamase. As a result, there are generations of β-lactam antibiotics that have improved efficacy and resistance to existing β-lactamases. However, pathogens have also developed further resistance mechanisms, mainly by obtaining new or altered β-lactamases. This is typified by the emergence of substrate-specific extended β-lactamases that inactivate many of the latest generations of cephalosporin and penicillin (but carbapenem does not apply to this) ² . As a result, the use of carbapenems such as imipenem and meropenem has increased significantly over the last 20 years. As expected, this increased consumption of carbapenem has led to the emergence of carbapenem-resistant gram-negative bacteria ³ . In particular, carbapenem-resistant enterobacteria (CRE) is a growing problem worldwide as evidenced by recent outbreaks in Chicago ⁵ and British Columbia ^{6 4} .

Carbapenemase β-lactamases, which inactivate carbapenems, can be divided into two categories based on the mechanism of hydrolysis of the β-lactam ring. The first category places serine residues in the active site that covalently attack the β-lactam ring, such as the KPC and OXA-48 types ⁷ . The second category is nucleophilic water that cleaves the ring, such as the Verona integron-coded metallo-β-lactamase (VIM) and New Delhi metallo-β-lactamase (NDM) types. It is a metallo β-lactamase (MBL) that uses a Zn ²⁺ atom to activate the molecule ⁸ . Several inhibitors of Ser β-lactamase are clinically available as co-drugs in which inhibitors are formulated with β-lactam antibiotics to overcome resistance (eg, clavulanic acid-amoxicillin, tazobactam- ⁹ ) Serbeta lactamase inhibitor abibactam, which is in phase III clinical trials in combination with piperacillin, sulbactam-ampicillin, and more recently various cephalosporins. Despite continued efforts ^10,11 , for practical and technical reasons, there is no comparable inhibitor of MBL in practice. First, until recently, MBL-derived CRE has not been considered as a major clinical problem, and this rapid increase has outpaced the development of MBL inhibitors. Second, the development of a single inhibitor that neutralizes clinically important MBL, such as VIM and NDM, is considered technically very difficult and crosses with human metalloenzymes. There is also concern overcoming the in vivo toxicity associated with reactivity. With the recent emergence of MBL as a significant clinical threat, powerful and safe inhibitors of MBL, particularly inhibitors against VIM and NDM, will be very beneficial in the management of infectious diseases.

Aspergillomarasmin A and B are fungal-derived molecules discovered and reported in the early 1960s ^12,13 . These molecules were used as inhibitors of angiotensin converting enzyme (ACE) in the 1980s ¹⁴ and in the early 1990s preclinical for inhibition of activation of human endothelin, a peptide that regulates vascular muscle contraction. ^{15 and 16 were} evaluated as candidate substances. From this previous study, AM-A was well tolerated and less toxic in mice (LD ₅₀ : 159.8 mg / kg (iv) compared to 28.5 mg / kg for EDTA), It has been demonstrated not to affect mean atrial blood pressure ¹⁷ . Another study reported that in rats, the LD ₅₀ was 250 mg / kg (iv) for AM-A and the LD ₅₀ was 660 mg / kg (iv) for AM-B. ¹⁴

Licomalathmin is also a fungal-derived molecule first reported in 1947 ¹⁸ .

  In this application, the compound Aspergilomarasmin A (AM-A) and certain analogs and derivatives are disclosed as potentiators of β-lactam antibiotics.

の化合物であって、式中、
Ｒ^１が、Ｃ（Ｏ）ＯＲ^５、Ｃ（Ｏ）ＮＨＲ^５、およびＣＨ（ＮＨ_２）Ｃ（Ｏ）ＯＲ^５から選択され；
Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５が、独立して、Ｈ、Ｃ_１−２４アルキル、Ｃ_１−６アルキレンＣ_６−１０アリール、Ｃ_１−６アルキレンＣ_３−１０シクロアルキル、Ｃ_１−６アルキレンＣ_３−１０ヘテロシクロアルキル、およびＣ_１−６アルキレン−ＯＣ（Ｏ）Ｃ_１−６アルキルから選択され；
ｎ、ｍ、およびｐが、１および２から独立して選択される；
化合物、またはその薬学的に許容可能な塩および／もしくは溶媒和物と
を含む医薬組成物であって、
１つまたは複数のβラクタム系化合物および１つまたは複数の式Ｉの化合物は、細菌感染症、または細菌感染症から生じる疾患、障害、もしくは状態を処置するために有効な量で存在する、
医薬組成物を含む。 Thus, in one embodiment, the present application is
One or more beta-lactam antibiotics;
One or more formulas I

A compound of the formula:
R ¹ is selected from C (O) OR ⁵ , C (O) NHR ⁵ , and CH (NH ₂ ) C (O) OR ⁵ ;
R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-24 alkyl, C _1-6 alkylene C _6-10 aryl, C _1-6 alkylene C _3-10 cycloalkyl, C Selected from _1-6 alkylene C _3-10 heterocycloalkyl, and C _1-6 alkylene-OC (O) C _1-6 alkyl;
n, m, and p are independently selected from 1 and 2;
A pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt and / or solvate thereof,
One or more β-lactam compounds and one or more compounds of formula I are present in an amount effective to treat a bacterial infection or a disease, disorder, or condition resulting from a bacterial infection,
Including a pharmaceutical composition.

の化合物であって、式中、
Ｒ^１が、Ｃ（Ｏ）ＯＲ^５、Ｃ（Ｏ）ＮＨＲ^５、およびＣＨ（ＮＨ_２）Ｃ（Ｏ）ＯＲ^５から選択され；
Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５が、独立して、Ｈ、Ｃ_１−２４アルキル、Ｃ_１−６アルキレンＣ_６−１０アリール、Ｃ_１−６アルキレンＣ_３−１０シクロアルキル、Ｃ_１−６アルキレンＣ_３−１０ヘテロシクロアルキル、およびＣ_１−６アルキレン−ＯＣ（Ｏ）Ｃ_１−６アルキルから選択され；
ｎ、ｍ、およびｐが、独立して１および２から選択される；
化合物、またはその薬学的に許容可能な塩および／もしくは溶媒和物の有効量と併用して１つまたは複数のβラクタム系抗生剤の有効量を、それを必要とする対象に投与することを含む、方法を含む。 In another embodiment, this application is a method of treating a bacterial infection, comprising one or more formula I

A compound of the formula:
R ¹ is selected from C (O) OR ⁵ , C (O) NHR ⁵ , and CH (NH ₂ ) C (O) OR ⁵ ;
R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-24 alkyl, C _1-6 alkylene C _6-10 aryl, C _1-6 alkylene C _3-10 cycloalkyl, C Selected from _1-6 alkylene C _3-10 heterocycloalkyl, and C _1-6 alkylene-OC (O) C _1-6 alkyl;
n, m, and p are independently selected from 1 and 2;
Administering to a subject in need thereof an effective amount of one or more beta-lactam antibiotics in combination with an effective amount of the compound, or a pharmaceutically acceptable salt and / or solvate thereof. Including methods.

  In another embodiment, this application is a method of treating or preventing a disease, disorder or condition resulting from a bacterial infection in a subject, comprising one or more compounds of formula I as defined above, or a pharmaceutical thereof Comprising administering to the subject an effective amount of one or more beta-lactam antibiotics in combination with an effective amount of an acceptable salt and / or solvate.

  In another embodiment, this application is a method of improving the efficacy of a β-lactam antibiotic for treating a bacterial infection, in combination with an antibiotic, one or more of formula I as defined above. A method comprising treating an effective amount of a compound, or a pharmaceutically acceptable salt and / or solvate thereof, with a subject in need thereof, and treating a disease, disorder or condition resulting from a bacterial infection A method of improving the efficacy of a β-lactam antibiotic, comprising combining one or more compounds of formula I as defined above, or a pharmaceutically acceptable salt thereof, and / or in combination with an antibiotic Including a method comprising administering an effective amount of a solvate to a subject in need thereof.

  The present application is also a method of treating a bacterial infection in an animal or human, wherein the predetermined β is used in combination with an amount of a pharmaceutically acceptable β-lactam antibiotic effective to treat the bacterial infection. Administering a lactamase inhibitor. The present application also provides a method of treating diseases associated with enterobacteria expressing metallo β-lactamase, particularly Klebsiella pneumoniae, in animals and human subjects, comprising an effective amount of a β-lactam antibiotic, particularly a carbapenem antibiotic In combination with a method comprising administering to a subject an effective amount of either AM-A, or any active AM-A analog or derivative.

  The present application also includes an antimicrobial combination comprising an effective amount of a fungal natural product, Aspergillomarasmin A or any active analog thereof, and a β-lactam antibiotic. In one embodiment, the fungal natural product is Aspergillomarasmin A or any active analog thereof and the β-lactam antibiotic is selected from the group consisting of penicillins, cephalosporins, monobactams and carbapenem antibiotics. . In a further embodiment, the fungal natural product is Aspergillomarasmin A or any active analog thereof and the β-lactam antibiotic is a carbapenem. In a further embodiment, the β-lactam antibiotic is meropenem.

  In an embodiment of the present application, the antimicrobial combination is formulated into a pharmaceutical dosage form. In a further embodiment of the present application, the antimicrobial combination is for use in a method of treating and / or preventing a bacterial infection in an individual, wherein the infection produces one or more beta-lactamases. Caused by bacteria, the method includes administering a formulation to a human or animal subject.

  In one embodiment, the bacterial infection is an infection of a bacterial expressing at least one metallo beta-lactamase (MBL), and the disease, disorder or condition resulting from the bacterial infection is an infection of a bacterial expressing at least one MBL Is a disease, disorder or condition resulting from.

  In one embodiment, the bacteria causing the infection are Staphylococcus aureus, Staphylococcus epidermidis and other coagulase negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactia, Enterococcus spp., Corynebacterium diphtherier, Listeria Selected from Monocytogenes, Bacillus anthracis, Neisseria meningitidis, Neisseria gonolet, Moraxella catarrhalis, Vibrio cholerae, and Campylobacter jejuni.

  In one embodiment, the bacteria causing the infection are enteric bacteria (including Escherichia coli, Salmonella, Klebsiella, Enterobacter), Pseudomonas aeruginosa, Acinetobacter, Haemophilus influenzae, Tetanus, Clostridium botulinum, Bacteroides , Prevoterra, Porphyromonas, Fusobacterium, Mycobacterium tuberculosis, and Leprosy.

  In one embodiment, the bacterium causing the infection is from the family Enterobacteriaceae.

  In one embodiment, the bacterium causing the infection is Klebsiella pneumonia.

  The present application is also a cell-based screening assay comprising a bacterial cell expressing a bacterial resistance gene, wherein the cell is (i) a gene encoding a protein that blocks entry of the molecule into the cell and (ii) from the cell Assays that have been modified to delete one or more of the genes that encode proteins that facilitate the excretion of the molecule.

The present application is also a method for identifying a compound that treats antibiotic resistance,
(A) contacting a bacterial cell expressing a bacterial resistance gene with one or more compounds, wherein the cell (i) a gene encoding a protein that blocks entry of the molecule into the cell and (ii) ) A protein that has been modified to delete one or more of the genes that encode proteins that promote the export of molecules from the cell, and wherein the one or more compounds are encoded by a bacterial resistance gene In contact with cells in the presence of antibiotics sensitive to
(B) identifying a compound that inhibits bacterial cell growth as a compound treating antibiotic resistance.

  Other features and advantages of the present application will become apparent from the following detailed description. However, the detailed description and specific examples, while representing examples of the present application, are given by way of illustration only and are not intended to limit the scope of the claims by these embodiments. As such, the widest interpretation without contradiction to the present specification is made.

  Embodiments of the present application will be described in more detail with reference to the following accompanying drawings.

AM-A (represented by (a)) is (b) NDM-1 (●) (IC ₅₀ : 4.0 ± 1.0 μM) and VIM-2 (◯) (IC ₅₀ : 9.6 ±). 2.4 μM), but the activity of OXA48 (■) was not affected by AM-A. (C) Removal of AM-A through the PD10 column does not restore NDM-1 activity, confirming irreversible inactivation. (D) The activity after inactivation is restored by adding excessive ZnSO ₄ . (E) The inactivation ratio of NDM-1 and VIM-2 can be saturated with [AM-A]. (F) ICP-MS confirms the deletion of Zn from NDM-1. Error bars represent the standard deviation of 3 iterations. (A, b) Not to carbapenem sensitive strain (b, E. coli BW25113 MIC = 0.008-0.016 μg / ml) but to CRE (a, K. pneumoniae N11-2218 MIC meropenem = 32 μg / ml) Microdilution checkerboard analysis showing the combined action of AM-A and meropenem selective. (C) Gram negative pathogens expressing VIM and NDM are sensitive to the meropenem / AM-A combination (2 μg / ml and 8 μg / ml, respectively) and isolates expressing AIM, IMP and SPM-1 Remained resistant. The results of treatment of CD-1 mice given a lethal dose of Klebsiella pneumoniae N11-2218 (meropenem, MIC: 32 μg / mL) by intraperitoneal injection are shown. (A, b) Groups of mice were treated with a single dose of meropenem (10 mg / kg), a combination of meropenem (10 mg / kg) + AM-A (10 mg / kg) or PBS by subcutaneous injection. Injections in the spleen (a) and liver (b) and bacterial load were determined by selective plating. Data are mean values with standard errors from two separate experiments. (C) Mice were treated with a single dose of meropenem (10 mg / kg), meropenem (10 mg / kg) + AM-A (30 mg / kg), or PBS by subcutaneous injection.

I. Definitions Unless defined otherwise, the definitions and embodiments described in this and other sections are used for all embodiments and aspects of the application herein described for proper understanding by those skilled in the art. It is intended to be possible.

  In understanding the scope of the present application, the term “comprising” and its derivatives as used herein refers to the presence of the described property, element, component, group, integer, and / or step. Is intended to be an open-ended term that does not exclude the presence of other characteristics, elements, components, groups, integers, and / or steps that are not described. The foregoing definitions also apply to terms that have similar meanings, such as terms such as “including”, “having” and their derivatives. As used herein, the term “consisting of” and its derivatives identify the presence of a described property, element, component, group, integer, and / or step, but a property, element, It is intended to be a closed term that excludes the presence of components, groups, integers, and / or steps. As used herein, the term “consisting essentially of” identifies the presence of the described property, element, component, group, integer, and / or step, and includes the property, element, component. It is not intended to significantly affect the basic and novel characteristics of elements, groups, integers, and / or steps.

  As used herein, terms such as “substantially”, “about”, and “approximate” are terms that have been modified so that the final result does not change significantly. Means a reasonable amount of deviation. The degree of these terms is constructed such that this deviation includes a deviation of at least ± 5% of the modified terms if they do not invalidate the meaning of the modified term.

  As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. For example, an embodiment comprising a “β-lactam antibiotic” is understood to represent a specific embodiment having one β-lactam antibiotic, or two or more additional β-lactam antibiotics.

  In embodiments including “additional” or “second” components, such as additional or second β-lactam antibiotics, the second component used herein is other Chemically different from the component or the first component. The “third” component is different from the other components, the first component, and the second component, and the listed or “additional” components are similarly different.

  The term “and / or” as used herein means that the listed items are presented or used individually or in combination. Indeed, the term means that “at least one” or “one or more” listed items are used or presented.

  The term “bacterial infection” as used herein refers to the invasion of cells or body tissues by exogenous undesirable bacteria. In one embodiment, the bacterial infection is an infection that expresses a metallo β-lactamase.

  As used herein, the term “infection that expresses a metallo β-lactamase” refers to the invasion of a cell or body tissue by a bacterium that expresses a metallo β-lactamase.

  The term “β-lactam antibiotic” as used herein refers to a class of antibiotics that have a β-lactam ring in the molecular structure. Examples include penicillin derivatives (penem series), cephalosporin (cephem series), monobactams and carbapenems. Most β-lactam antibiotics act by inhibiting the biosynthesis of bacterial cell walls and are the most widely used group of antibiotics.

The term “alkyl” as used herein, whether alone or as part of another group, means a straight or branched chain hydrocarbon group. For example, the term _C1-24 alkyl is ₁ , 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, ₁₃ , 14, ₁₅ , 16, _{17, 18} , 19, 20, 21, Means an alkyl group having 22, 23, or 24 carbon molecules;

The term “alkylene” as used herein refers to a straight or branched chain saturated hydrocarbon group, a saturated carbon chain containing substituents at two of the ends. For example, the term C _1-6 alkylene means an alkylene group having 1, 2, 3, 4, 5, or 6 carbon atoms.

The term “cycloalkyl” as used herein, means a saturated alkyl group having at least one ring, whether used alone or as part of another group. For example, the term C _3-10 cycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

  The term “aryl” or “aromatic” as used herein, whether used alone or as part of another group, refers to a ring comprising at least one aromatic ring. In one embodiment of the present application, the aryl group comprises 6 or 10 (carbon atoms) such as phenyl, naphthyl, or indanyl.

The term “heterocycloalkyl” as used herein, whether used alone or as part of another group, contains 5, 6, 7, 8, 9, or 10 atoms. Refers to a non-aromatic monocyclic or polycyclic system, wherein one or more of these atoms, such as 1-6, 1-5, 1-4, or 1-3, are O, S, A hetero moiety selected from NH and NC _1-6 alkyl, with the remaining atoms being C, CH, or CH ₂ . Heterocycloalkyl groups are either saturated or unsaturated (ie, contain one or more double bonds) and may contain more than one ring. If the heterocycloalkyl group contains more than one ring, the rings may be linked by condensation, bridges, spiro bonds or single bonds.

  A first cyclic group “fused” with a second cyclic group means that the first ring and the second ring share at least two adjacent atoms therebetween.

  A first cyclic group that is “bridged” with a second cyclic group means that the first ring and the second ring share at least two non-adjacent atoms therebetween.

  A first cyclic group “spiro-bonded” with a second cyclic group means that the first and second rings share one atom therebetween.

の化合物を指す。 As used herein, the term “aspergillomarasmine A” or “AM-A” is a compound of the formula

Of the compound.

の化合物を指す。 As used herein, the term “aspergillomarasmine B” or “AM-B” is a compound of the formula

Of the compound.

の化合物を指す。 As used herein, the term “licomalasmin” has the formula

Of the compound.

  The term “pharmaceutically acceptable salt” means an acid addition salt or a base addition salt that is suitable for or suitable for treatment of a subject.

  The term “pharmaceutically acceptable salts” includes salts commonly used to form addition salts of free acids or free bases. The nature of the salt is not critical as long as it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts are prepared from inorganic or organic acids. Examples of such inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Examples of suitable organic acids include, for example, aliphatic acids, cycloaliphatic acids, aromatic acids, aryl fatty acids, heterocyclic acids, carboxylic acids, and organic acids of the sulfonic acid class, such as , But not limited to, formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, maleic acid, embonic acid (pamo acid) methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, pantothenic acid , Benzenesulfonic acid, toluenesulfonic acid, sulfanilic acid, mesylic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, malonic acid, galactic acid, and galacturonic acid . Suitable pharmaceutically acceptable base addition salts include, but are not limited to, metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or N, N′-dibenzylethylenediamine, Examples include organic salts made from chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine and procaine.

  Formation of the desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the salt formed is isolated by filtration, extraction or any other suitable method.

  The term “solvate” as used herein refers to a complex formed between a compound and a solvent from which the compound is precipitated or from which the compound is made. . Thus, the term “solvate” as used herein means a compound, or salt compound, in which molecules of a suitable solvent are incorporated into the crystal lattice. Examples of suitable solvents include ethanol, water and the like. When water is the solvent, the molecule is called a “hydrate”. The formation of solvates varies depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. In general, solvates are dried under atmospheric conditions and become an azeotrope. Selection of appropriate conditions for forming a particular solvate can be made by one skilled in the art.

  The term “pharmaceutically acceptable solvate” means a solvate that is suitable for or suitable for treatment of a subject. In pharmaceutically acceptable solvates, a suitable solvent is physiologically tolerable at the dosage used or administered.

  As used herein, the expression “disease, disorder, or condition resulting from a bacterial infection” refers to any disease, disorder, or condition that occurs directly or indirectly in a subject due to the presence of a bacterial infection. Point to.

  As used herein, the term “subject” includes all animals of the animal kingdom including mammals.

  The term “pharmaceutical composition” as used herein refers to a composition of interest for pharmaceutical use.

  The term “pharmaceutically acceptable” means compatible with the treatment of subjects such as horses and mammals such as humans.

  As used herein, the term “parenteral” means taken into the body or administered by a method other than administration through the gastrointestinal tract.

  As used herein, the term “administered” refers to an effective amount of an antibiotic and a compound comprising a compound of formula I or a salt and / or solvate thereof, either in a cell culture or subject. Means administration to the cells.

  The term “effective amount” or “therapeutically effective amount” as used herein means an amount that is effective between the dose and time required to achieve the desired result. For example, in view of treating a bacterial infection, or a disease, disorder, or condition resulting from a bacterial infection, an effective amount of an antibiotic and / or a compound of formula I or a salt and / or solvate thereof is, for example, an antibiotic An amount that reduces bacterial infection compared to a bacterial infection that has not been administered an agent and / or a compound of formula I or a salt and / or solvate thereof. Further, in view of improving the efficacy of antibiotics for the treatment of bacterial infections or diseases, disorders, or conditions resulting from bacterial infections, an effective amount of a compound of formula I, or a salt and / or solvate thereof, is For example, an amount that reduces bacterial infection compared to reducing bacterial infection with administration of antibiotics alone. The phrase “reduce infection” means, for example, reducing the amount of an infectious agent in a subject and / or reducing symptoms of an infection. An effective amount may vary depending on factors such as the disease state, age, sex, and / or weight of the subject. The amount of a given compound or composition corresponding to such an amount will depend on factors such as the given compound or composition, pharmaceutical formulation, route of administration, condition, disease or disorder type, the particularity of the subject being treated, etc. Although it will vary, this can be determined by routine procedures for those skilled in the art.

  As used herein, the terms “to treat”, “treat”, and “treatment” refer to techniques for obtaining beneficial or desired results, including clinical results. Beneficial or desirable clinical consequences include, but are not limited to, a reduction in the scope of bacterial infections, whether partial or complete, detectable or undetectable, Stable condition (ie, not exacerbated), preventing the spread of bacterial infections, slowing or slowing the progression of infections, restoring or ameliorating the state of bacterial infections, reducing the recurrence of bacterial infections, bacterial infections Reduction, stabilization, reduction or recovery of one or more diseases, disorders or conditions resulting from, reduction of recurrence of one or more diseases, disorders or conditions resulting from bacterial infections, and bacterial infections and / or bacterial infections Remission of one or more symptoms or conditions resulting from the disease. “To treat”, “treat” and “treatment” also mean an extended survival time compared to the expected survival time when not receiving treatment. As used herein, “to treat”, “treat”, and “treatment” also mean prophylactic treatment. For example, patients with an early bacterial infection are treated to prevent progression, or alternatively, remission subjects are treated to prevent recurrence.

  “Relief” of an infectious disease, disease, disorder, and / or condition is a range of infectious disease, disease, disorder, and / or condition and / or undesirable clinical symptoms, and these infectious diseases, diseases, disorders, and / or conditions It means less than if the condition has not been treated and / or a change in progression over time is delayed or prolonged.

  As used herein, the terms “prevention or prophylaxis” and the like refer to a subject suffering from a bacterial infection and / or a disease, disorder and / or condition resulting from a bacterial infection, or a bacterial infection and Or to reduce the risk or likelihood of presenting symptoms associated with a disease, disorder and / or condition resulting from a bacterial infection.

  For example, when used in connection with the treatments, uses, compositions and kits of the present application, a subject such as, for example, a “necessary” subject, is a subject diagnosed with a bacterial infection or a disease, disorder, or condition resulting from a bacterial infection A subject suspected of having, having been in contact with, and / or having previously received these treatments.

II. Methods and Applications Cell-based screening for inhibitors of NDM-1 metallo β-lactamase was performed using a collection of natural product extracts from environmental microorganisms. In order to increase the sensitivity of screening to the discovery of MBL inhibitors, a test strain of E. coli BW25113 (E. coli BW25113ΔbamBΔtolC) in which the genes for bamB and tolC are independently deleted was generated. BamB is involved in the outer membrane porin assembly and when broken it increases permeability to small molecules ¹⁹ , and TolC is a 3 molecule such as AcrA-AcrB-TolC that actively excludes small molecules from cells. which is part of the small molecule emission system ^20. Thus, E.I. E. coli BW25113ΔbamBΔtolC increases the sensitivity of screening for “hit” discovery. This strain was transformed into the chromosomal [E. bla _NDM-1 β-lactamase gene under the control of the pLac promoter. E. coli BW25113ΔbamBΔtolCΔaraDAB :: pLac (bla _NDM-1 )] was further modified. This strain was screened in the presence of a sublethal concentration of meropenem combined with ˜500 natural product extracts (1/4 of a minimum inhibitory concentration of 0.125 μg / ml).

  In an exemplary embodiment of the present application, the screening involves an E. coli extract from an Aspergillus versicolol strain (identified by 18S rRNA gene sequence). Reproducible hits were obtained with excellent properties that restore the activity of the meropenem antibiotic against E. coli screening strains. The selectivity of the extract for neutralizing NDM-1 activity in vivo was confirmed using the carbapenem meropenem using the purified enzyme and the chromogenic β-lactamase substrate nitrocefin. A. Activity as index From the purification of the active compound from the Versiccolor fermentation broth and subsequent detailed chemical characterization, the MBL inhibitor was identified as Aspergillomarasmin A (AM-A).

の化合物であって、式中
Ｒ^１が、Ｃ（Ｏ）ＯＲ^５、Ｃ（Ｏ）ＮＨＲ^５、およびＣＨ（ＮＨ_２）Ｃ（Ｏ）ＯＲ^５から選択され；
Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５が、独立して、Ｈ、Ｃ_１−２４アルキル、Ｃ_１−６アルキレンＣ_６−１０アリール、Ｃ_１−６アルキレンＣ_３−１０シクロアルキル、Ｃ_１−６アルキレンＣ_３−１０ヘテロシクロアルキル、およびＣ_１−６アルキレン−ＯＣ（Ｏ）Ｃ_１−６アルキルから選択され；
ｎ、ｍ、およびｐが、独立して１および２から選択される；
式Ｉの化合物またはその薬学的に許容可能な塩および／もしくは溶媒和物の有効量と併用して１つまたは複数のβラクタム系抗生剤の有効量を、その必要のある対象に投与することを含む、方法を含む。 Accordingly, in one embodiment, the present application is a method of treating a bacterial infection, comprising one or more formula I

Wherein R ¹ is selected from C (O) OR ⁵ , C (O) NHR ⁵ , and CH (NH ₂ ) C (O) OR ⁵ ;
R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-24 alkyl, C _1-6 alkylene C _6-10 aryl, C _1-6 alkylene C _3-10 cycloalkyl, C Selected from _1-6 alkylene C _3-10 heterocycloalkyl, and C _1-6 alkylene-OC (O) C _1-6 alkyl;
n, m, and p are independently selected from 1 and 2;
Administering to a subject in need thereof an effective amount of one or more beta-lactam antibiotics in combination with an effective amount of a compound of formula I, or a pharmaceutically acceptable salt and / or solvate thereof. Including a method.

  This application also relates to β-lactams in combination with one or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof, for treating bacterial infections in a subject. The use of a series antibiotic, one or more compounds of formula I as defined above, or a pharmaceutically acceptable salt thereof and / or for the preparation of a medicament for treating a bacterial infection in a subject Use of a β-lactam antibiotic in combination with a solvate, as well as a beta-lactam antibiotic and one or more of the above defined formula I for use in treating a bacterial infection in a subject A compound, or a pharmaceutically acceptable salt and / or solvate thereof.

  In another embodiment, this application is a method of treating or preventing a disease, disorder, or condition resulting from a bacterial infection in a subject, comprising one or more compounds of formula I as defined above or a pharmaceutical thereof Comprising administering to the subject an effective amount of one or more beta-lactam antibiotics in combination with an effective amount of an acceptable salt and / or solvate.

  This application also provides one or more compounds of formula I as defined above or a pharmaceutically acceptable salt and / or solvent thereof for treating a disease, disorder, or condition resulting from a bacterial infection in a subject. Use of a β-lactam antibiotic in combination with a Japanese one, one or more of the formulas I as defined above for preparing a medicament for treating a disease, disorder or condition resulting from a bacterial infection in a subject Of β-lactam antibiotics in combination with a compound of the invention or a pharmaceutically acceptable salt and / or solvate thereof, and used to treat a disease, disorder or condition resulting from a bacterial infection in a subject Β-lactam antibiotics and one or more compounds of formula I as defined above or pharmaceutically acceptable salts and / or solvates thereof for .

  In another embodiment, this application is a method of improving the efficacy of a β-lactam antibiotic for treating a bacterial infection, wherein the formula is used in combination with one or more of the above defined formulas. Comprising administering an effective amount of a compound of I, or a pharmaceutically acceptable salt and / or solvate thereof, to a subject in need thereof.

  This application also provides one or more compounds of formula I as defined above, or pharmaceutically acceptable thereof, for improving the efficacy of beta-lactam antibiotics for treating bacterial infections in a subject. Use of a salt and / or solvate, one or more of the formula I as defined above for preparing a drug that improves the efficacy of a β-lactam antibiotic for treating a bacterial infection in a subject One or more of the use of the compound, or a pharmaceutically acceptable salt and / or solvate thereof, and to improve the efficacy of a beta-lactam antibiotic to treat a bacterial infection in a subject Of the formula I as defined above, or a pharmaceutically acceptable salt and / or solvate thereof.

  The application also provides a method for improving the efficacy of a β-lactam antibiotic for treating a disease, disorder, or condition resulting from a bacterial infection, comprising one or more compounds of formula I as defined above, or Including administering an effective amount of a pharmaceutically acceptable salt and / or solvate to a subject in need thereof.

  The application also provides one or more compounds of formula I as defined above, or a compound thereof, for improving the efficacy of a β-lactam antibiotic for treating a disease, disorder, or condition resulting from a subject bacterial infection. The use of pharmaceutically acceptable salts and / or solvates, to prepare drugs that improve the efficacy of β-lactam antibiotics for treating diseases, disorders or conditions resulting from bacterial infections in a subject, Use of one or more compounds of formula I as defined above, or a pharmaceutically acceptable salt and / or solvate thereof, and treating a disease, disorder or condition resulting from a bacterial infection in a subject One or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solutions thereof, used to improve the efficacy of β-lactam antibiotics Including the use of hydrates.

  In one embodiment, the bacterial infection is an infection of a bacteria that expresses at least one metallo β-lactamase (MBL), and the disease, disorder, or condition resulting from the bacterial infection is a bacteria that expresses at least one MBL. Is a disease, disorder, or condition that results from an infection. In one embodiment, the MBL is IMP-type, Verona integron-coded metallo β-lactamase (VIM), or New Delhi metallo β-lactamase (NDM). In a further embodiment, the MBL is VIM or NDM.

  In one embodiment, the bacterial infection is an infection of at least one carbapenem resistant gram negative bacterium.

  In one embodiment, the bacterial infection is an infection of at least one bacterial belonging to the genus Acinetobacter, Pseudomonas of the Enterobacteriaceae family.

  In one embodiment, the enteric bacterium is Klebsiella or E. coli, such as Klebsiella pneumonia. In another embodiment, the Pseudomonas bacterium is Pseudomonas aeruginosa.

  In one embodiment, the bacteria causing the infection are Staphylococcus aureus, Staphylococcus epidermidis and other coagulase negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactia, Enterococcus spp., Corynebacterium diphtherier, Listeria Selected from Monocytogenes, Bacillus anthracis, Neisseria meningitidis, Neisseria gonolet, Moraxella catarrhalis, Vibrio cholerae, and Campylobacter jejuni.

  In one embodiment, the bacteria causing the infection are Enterobacteriaceae (including Escherichia coli, Salmonella, Klebsiella, Enterobacter), Pseudomonas aeruginosa, Acinetobacter, Haemophilus influenzae, Tetanus, Clostridium botulinum, Bacteroides Selected from the genera, Prevotella, Porphyromonas, Fusobacterium, Mycobacterium tuberculosis, and Leprosy.

  In one embodiment, the bacterium causing the infection is from the family Enterobacteriaceae.

  In one embodiment, the bacterium causing the infection is Klebsiella pneumoniae.

  A disease, disorder, or condition resulting from a bacterial infection includes all etiologies common to bacterial infections that express MBL. These are well known to those skilled in the art. Some of the more general examples are listed below for the better known MBL expressing bacteria, but these listings are not exhaustive and are listed for one MBL expressing bacterium. It will be apparent to those skilled in the art that many of the diseases, disorders, and conditions are common to other MBL-expressing bacteria.

  In one embodiment, the disease, disorder, or condition resulting from a bacterial infection, such as a Klebsiella pneumoniae infection, includes, but is not limited to, pneumonia (eg bronchial pneumonia or bronchitis), thrombophlebitis, urinary tract Infection (UTI), cholecystitis, diarrhea, upper respiratory tract infection, lower biliary tract infection, wound infection, surgical wound infection, osteomyelitis, meningitis, bacteremia, septicemia (septicemia, sepsis) ), Septic shock, nasal cirrhosis, rhinorrhea, ankylosing spondylitis, destructive changes in human lungs through inflammation and hemorrhage with cell death (necrosis), lung abscess, cavitation, empyema or ural adhesion adhesions), or a combination thereof.

  In one embodiment, the disease, disorder, or condition resulting from a bacterial infection, such as a Pseudomonas aeruginosa infection, includes but is not limited to cystic fibrosis, pneumonia, bacteremia, endocarditis, marrow Membrane inflammation, brain tumor, septic shock, UTI, gastrointestinal infection (eg, diarrhea, enteritis, or total enteritis), skin infection (eg, gangrene abscess), soft tissue infection, burn infection, outer ear infection Bacterial keratitis, endophthalmitis, infections due to the presence of medical devices, hospitalized infections, infections caused by low-quality water, postoperative infections, or osteomyelitis, or combinations thereof.

  In one embodiment, the disease, disorder, or condition resulting from a bacterial infection, such as an infection of E. coli, includes, but is not limited to, an intestinal infection (eg, diarrhea), an intraperitoneal infection, cholecystitis, In bacteremia, cholangitis, UTI, meningitis, pneumonia, purulent arthritis, endophthalmitis, purulent thyroiditis, osteomyelitis, endocarditis, skin infection or soft tissue infection, or combinations thereof is there.

  In one embodiment, the subject is a human. In a further embodiment, the subject is an animal such as a companion animal or livestock.

  The one or more antibiotics are selected from any antibiotics that treat bacterial infections that express β-lactamase. In one embodiment, the one or more antibiotics are beta-lactam antibiotics. In one embodiment, the β-lactam antibiotic is selected from penicillin derivatives (penems), cephalosporins (cephems), monobactams, and carbapenems. In one embodiment, the beta-lactam antibiotics are imipenem, ertapenem, meropenem, doripenem, biapenem, panipenem, ticarcillin, ampicillin, amoxicillin, carbenicillin, piperacillin, azurocillin, medulocillin, ticofetradim, cefotaxime, Selected from.

  In another embodiment, the one or more antibiotics are carbapenem antibiotics. In one embodiment, the carbapenem antibiotic is selected from meropenem, biapenem, doripenem, ertapenem, panipenem, and imipenem.

相対立体化学を有する。 In one embodiment, the compound of formula I is:

Has relative stereochemistry.

In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are each H. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are independently C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC. (O) Selected from C _1-6 alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same and are C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC. (O) Selected from C _1-6 alkyl.

In one embodiment, when at least one of R ² , R ³ , R ⁴ , and R ⁵ is other than H, the compound of formula I is R ² , R ³ , R ⁴ , and R ⁵ are each H A prodrug of the active compound.

相対立体化学を有する。 In one embodiment, R ¹ is CH (NH ₂ ) C (O) OR ⁵ and R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C _{1 -4} alkylene _{C 5-6} heterocycloalkyl, and _{C 1-4} is selected from alkylene -OC _{(O) C 1-6} alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same. In one embodiment, when R ¹ is CH (NH ₂ ) C (O) OR ⁵ , the compound of formula I is:

Has relative stereochemistry.

In one embodiment, R ¹ is C (O) OR ⁵ and R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C _1-4 alkylene C _{5- 6} heterocycloalkyl, and _{C 1-4} is selected from alkylene -OC _{(O) C 1-6} alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same.

In one embodiment, R ¹ is C (O) NHR ⁵ and R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C _1-4 alkylene C _{5. -6} heterocycloalkyl, and _{C 1-4} is selected from alkylene -OC _{(O) C 1-6} alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same.

である。 In one embodiment, heterocycloalkyl is

It is.

  In one embodiment, n, m, and p are each 1.

  In one embodiment, the compound of formula I is selected from AM-A, AM-B, and licomalasmin or pharmaceutically acceptable salts and / or solvates thereof. In a further embodiment, the compounds of formula I are prodrugs of AM-A, AM-B, and licomalasmin or pharmaceutically acceptable salts and / or solvates thereof.

Compounds of formula I are available via isolation from fungal sources using known natural product isolation methods or by chemical synthesis using methods known to those skilled in the art. Prepared. For example, compounds of formula I wherein R ² , R ³ , R ⁴ , and / or R ⁵ are other than H can be obtained using standard esterification or amidation methods using R ² , R ³ , R ^4. And / or from compounds of formula I wherein R ⁵ is H.

  Antibiotics are administered to or used in subjects in a variety of forms depending on the chosen route of administration, as will be appreciated by those skilled in the art. In one embodiment, the antibiotic is administered orally (including sublingual and buccal) or parenteral (intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, Rectal, topical, patch, pump, and transdermal) administration or use to a subject, and antibiotics are formulated accordingly. Conventional techniques and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington's Pharmaceutical Sciences (2000-20th edition) and The United States Pharmaformia: The National Formula 24 (1999). ). In general, antibiotics are used and administered to a subject in the form that is available. Such forms include, for example, their pharmaceutically acceptable salt forms, microparticulate forms of zwitterions and injectable or infusible suspension forms.

  A compound of formula I, or a pharmaceutically acceptable salt and / or solvate thereof, can also be administered to or used in a subject in a variety of forms depending on the chosen route of administration, as will be understood by those skilled in the art. The In one embodiment, the compound of formula I is administered orally (including sublingual and buccal) or parenteral (intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal). (Including rectal, patch, pump, and transdermal) administration to or use in subjects, and compounds, salts and / or solvates are formulated to follow. Again, conventional techniques and ingredients for the selection and preparation of suitable compositions are described, for example, by Remington's Pharmaceutical Sciences (2000-20th edition), and The United States Pharmacopeia: The National Nutrition: USP 24 NF19).

  Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form is sterile and is fluid to the extent that easy syringability exists.

  In one embodiment, parenteral administration is by continuous administration for a selected time. Solutions suitable for parenteral administration are prepared by methods known to those skilled in the art. For example, antibiotics or compounds of formula I, or salts and / or solvates thereof, are prepared with water, optionally mixed with a surfactant such as hydroxypropylcellulose. Dispersions are also prepared with or without alcohol in glycerol, liquid polyethylene glycol, DMSO and mixtures thereof, and in oil. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

  Compositions for nasal administration are conveniently formulated as aerosols, drops, gels or powders. Aerosol formulations generally include a solution or fine suspension of the active agent in a physiologically acceptable aqueous or non-aqueous solvent and are usually present in a sealed container in sterile form in single or multiple doses This takes the form of a cartridge or refill for use with a spray device. Alternatively, the sealed container is a single dosing device, such as a single dose nasal inhaler or aerosol dispenser fitted with a metering valve intended to be disposed of after use. Where the dosage form includes an aerosol dispenser, this includes a propellant, for example a compressed gas such as compressed air or an organic propellant such as a fluorochlorohydrocarbon. In one embodiment, the aerosol dosage form takes the form of a pump atomizer.

  Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, gum arabic, tragacanth, gelatin and / or glycerin. . Compositions for rectal administration are in the form of convenient suppositories containing a conventional suppository base such as cocoa butter.

  In another embodiment, the antibiotic, or compound of Formula I, or salt and / or solvate thereof is administered orally, for example with an inert diluent or an assimilable edible carrier, or hard shell or soft shell gelatin. Encapsulated in capsules, compressed into tablets, or incorporated directly into food. For oral administration, an antibiotic, or a compound of formula I, or a salt and / or solvate thereof, is incorporated into an excipient, eg, an ingestible tablet, buccal tablet, troche, capsule, elixir, suspension Used in the form of agents, syrups, wafers, etc. Oral dosage forms also include modified release formulations such as, for example, immediate release and timed-release formulations. Examples of modified release formulations include, for example, sustained release (SR), extended-release (ER, XR, or XL), time-release or timed-release, controlled-release (CR ) Type, or continuous release (CR or Contin) type, coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, agglomerated particles such as molecular sieve type particles, or micro-hollow permeation It is used in the form of a fiber bundle or a cut-type hollow osmotic fiber aggregated or held in a fibrous packet. In one embodiment, the sustained release composition is formulated as a liposome, or the active compound is formulated to be protected with a differently degraded coating, such as by microencapsulation, multiple coatings, and the like. Liposome delivery systems include, for example, small unilamellar liposomes, large unilamellar liposomes, and multilamellar liposomes. In one embodiment, liposomes are formed from various lipids such as cholesterol, stearylamine, or phosphatidylcholine.

  It is also possible to lyophilize the antibiotic or the compound of formula I, or a salt and / or solvate thereof, and use the resulting lyophilizate, for example for the preparation of a product for injection. .

  In one embodiment, an antibiotic agent, or a compound of formula I, or a salt and / or solvate thereof, is coupled with a soluble polymer as a tabletable drug carrier. Such polymers include, for example, polyvinyl pyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. In further embodiments, the antibiotic or the compound of formula I, or a salt and / or solvate thereof, for example, polylactic acid, polyglycolic acid, polylactic acid and polyglycolic acid copolymer, polyepsilon caprolactone, polyhydroxybutanoic acid, Coupling with biodegradable classes of polymers useful in achieving sustained drug release, such as polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogel cross-linked or amphiphilic block copolymers .

  Antibiotics and compounds of formula I, or salts and / or solvates thereof, are used in combination with each other. Antibiotics and compounds of formula I, or salts and / or solvates thereof, are used or administered at different times and / or in different modes of administration (ie different routes of administration) or together in the same pharmaceutical preparation. Be administered.

  In one embodiment, the antibiotic and the compound of formula I, or salts and / or solvates thereof, are used or administered at separate times and / or by the route of administration. For example, an antibiotic is administered by injection and a compound of formula I, or a salt and / or solvate thereof, is administered orally. In another example, the antibiotic is administered orally and the compound of formula I, or a salt and / or solvate thereof, is administered by injection. In a further example, both the antibiotic and the compound of formula I, or a salt and / or solvate thereof, are administered by injection. When the antibiotic and the compound of formula I, or a salt and / or solvate thereof, are administered at separate times and / or by the route of administration, the antibiotic is the compound of formula I, or a salt and / or solvent thereof It is administered or used either before or after use.

  In another embodiment, the antibiotic and the compound of formula I, or a salt and / or solvate thereof, are administered simultaneously. As used herein, “simultaneous administration” of two substances to a subject refers to an antibiotic and a compound of formula I, or a salt and / or solvate thereof, both of which are simultaneously administered to the subject. It is meant to be provided so as to be biologically active therein. The exact details of administration will depend on the pharmacokinetics of the antibiotic and the compound of formula I, or salts and / or solvates thereof, in the presence of each other, and the antibiotic and the compound of formula I or its Salts and / or solvates are administered within a few hours of each other, and if pharmacokinetics are appropriate, antibiotics and compounds of formula I, or salts and / or solvates thereof, Administration within a range of 24 hours or more from one administration may be included. The design of an appropriate dosing regimen is within the scope of those skilled in the art.

  In one embodiment, the antibiotic and the compound of formula I, or salts and / or solvates thereof, are administered to the subject in a single composition or formulation.

  In another embodiment of the present application, the antibiotic and the compound of formula I, or salts and / or solvates thereof, are administered to the subject in a non-concurrent manner.

  In further embodiments of the present application, the antibiotic and the compound of formula I, or salts and / or solvates thereof, are administered to the subject in a simultaneous format and then administered to the subject in a non-simultaneous format, or simultaneously. It is administered alternately in a format that is not simultaneous with the format.

  The method of treatment includes administering to the subject an antibiotic and a compound of formula I, or a salt and / or solvate thereof, optionally consisting of a single dose or alternatively comprising a series of doses. . The length of the treatment period depends on the severity of the disease, disorder, or condition, the age of the subject, the antibiotic, and the compound of formula I, or a salt and / or solvate thereof, the antibiotic, and the compound of formula I , Or the activity of its salts and / or solvates, and / or combinations thereof.

  In some embodiments, the antibiotic is administered or treated according to treatment protocols known for antibiotics in treating bacterial infections.

  In one embodiment, the antibiotic and the compound of formula I, or salts and / or solvates thereof, are administered or used as soon as possible after exposure to the bacteria. In one embodiment, the antibiotic, and the compound of Formula I, or salt and / or solvate thereof, until a treatment for a bacterial infection is achieved, for example, until complete eradication of the bacteria is achieved, or It is administered or used until the subject's biodefense is no longer overwhelmed and the number of bacteria is reduced to the point where the remaining bacteria can be killed.

  Antibiotics, and compounds of formula I, or salts and / or salt doses thereof, are determined according to their pharmacodynamic properties, mode of administration, subject age, health status and weight, nature and degree of symptoms, frequency of treatment and If present, it will vary depending on the type of combination therapy and the clearance ratio in the subject being treated. One of ordinary skill in the art can determine the appropriate dose based on the above factors. Antibiotics, and compounds of Formula I, or salts and / or solvates thereof, may be initially administered at a suitable dose that can be adjusted to the required amount depending on the clinical response.

  In one embodiment, the antibiotic dose is less than or equal to the dose of the drug when used alone. Such doses are known to those skilled in the art or are readily determined by those skilled in the art.

の化合物であって、式中、
Ｒ^１が、Ｃ（Ｏ）ＯＲ^５、Ｃ（Ｏ）ＮＨＲ^５、およびＣＨ（ＮＨ_２）Ｃ（Ｏ）ＯＲ^５から選択され；
Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５が、独立して、Ｈ、Ｃ_１−２４アルキル、Ｃ_１−６アルキレンＣ_６−１０アリール、Ｃ_１−６アルキレンＣ_３−１０シクロアルキル、Ｃ_１−６アルキレンＣ_３−１０ヘテロシクロアルキル、およびＣ_１−６アルキレン−ＯＣ（Ｏ）Ｃ_１−６アルキルから選択され；
ｎ、ｍ、およびｐが、独立して１および２から選択される；
式Ｉの化合物、またはその薬学的に許容可能な塩および／または溶媒和物と
を含む医薬組成物であって、
１つまたは複数のβラクタム系抗生剤および１つまたは複数の式１の化合物が、細菌感染症、または細菌感染症から生じる疾患、障害、もしくは状態を処置するために有効な量で存在する、
医薬組成物を含む。 III. Compositions and kits of the present application
One or more beta-lactam antibiotics;
One or more formulas I

A compound of the formula:
R ¹ is selected from C (O) OR ⁵ , C (O) NHR ⁵ , and CH (NH ₂ ) C (O) OR ⁵ ;
R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-24 alkyl, C _1-6 alkylene C _6-10 aryl, C _1-6 alkylene C _3-10 cycloalkyl, C Selected from _1-6 alkylene C _3-10 heterocycloalkyl, and C _1-6 alkylene-OC (O) C _1-6 alkyl;
n, m, and p are independently selected from 1 and 2;
A pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt and / or solvate thereof,
One or more beta-lactam antibiotics and one or more compounds of Formula 1 are present in an amount effective to treat a bacterial infection, or a disease, disorder, or condition resulting from a bacterial infection,
Including a pharmaceutical composition.

This application also
One or more beta-lactam antibiotics;
A pharmaceutical composition comprising one or more compounds of formula I as defined above, or a pharmaceutically acceptable salt and / or solvate thereof,
The efficacy of one or more beta-lactam antibiotics and one or more compounds of formula I for the treatment of a bacterial infection or a disease, disorder or condition resulting from a bacterial infection Present in an effective amount to improve
Including a pharmaceutical composition.

の化合物であって、式中、
Ｒ^１が、Ｃ（Ｏ）ＯＲ^５、Ｃ（Ｏ）ＮＨＲ^５、およびＣＨ（ＮＨ_２）Ｃ（Ｏ）ＯＲ^５から選択され；
Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５が、独立して、Ｈ、Ｃ_１−２４アルキル、Ｃ_１−６アルキレンＣ_６−１０アリール、Ｃ_１−６アルキレンＣ_３−１０シクロアルキル、Ｃ_１−６アルキレンＣ_３−１０ヘテロシクロアルキル、およびＣ_１−６アルキレン−ＯＣ（Ｏ）Ｃ_１−６アルキルから選択され；
ｎ、ｍ、およびｐが、独立して１および２から選択される；
式Ｉの化合物またはその薬学的に許容可能な塩および／もしくは溶媒和物と、
任意に、１つまたは複数のβラクタム系抗生剤ならびに１つまたは複数の式Ｉの化合物またはその薬学的に許容可能な塩および／もしくは溶媒和物を、それを必要とする対象に投与するための説明書と
を含む、キットを含む。 This application is also a kit for the treatment of bacterial infections, or diseases, disorders, or conditions resulting from bacterial infections,
One or more beta-lactam antibiotics;
One or more formulas I

A compound of the formula:
R ¹ is selected from C (O) OR ⁵ , C (O) NHR ⁵ , and CH (NH ₂ ) C (O) OR ⁵ ;
R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-24 alkyl, C _1-6 alkylene C _6-10 aryl, C _1-6 alkylene C _3-10 cycloalkyl, C Selected from _1-6 alkylene C _3-10 heterocycloalkyl, and C _1-6 alkylene-OC (O) C _1-6 alkyl;
n, m, and p are independently selected from 1 and 2;
A compound of formula I or a pharmaceutically acceptable salt and / or solvate thereof;
Optionally, for administering one or more beta-lactam antibiotics and one or more compounds of formula I or pharmaceutically acceptable salts and / or solvates thereof to a subject in need thereof. Including kits.

The present application is also a kit for treating a bacterial infection, or a disease, disorder, or condition resulting from a bacterial infection,
One or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof,
One or more compounds of formula I or pharmaceutically acceptable salts and / or solvates thereof are administered a bacterial infection or an antibiotic for a disease, disorder or condition resulting from a bacterial infection And a kit containing instructions for administration to a subject.

The present application is also a kit for improving the efficacy of β-lactam antibiotics for the treatment of bacterial infections or diseases, disorders or conditions resulting from bacterial infections,
One or more beta-lactam antibiotics;
One or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof,
Optionally, one or more beta-lactam antibiotics and one or more compounds of formula I, or pharmaceutically acceptable salts and / or solvates thereof, are administered to a subject in need thereof. Including kits, including instructions for.

The present application is also a kit for improving the efficacy of β-lactam antibiotics for the treatment of bacterial infections, or diseases, disorders, or conditions resulting from bacterial infections,
One or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof,
One or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof, to treat a bacterial infection or a disease, disorder or condition resulting from a bacterial infection And a kit containing instructions for administering to a subject being administered a β-lactam antibiotic.

  The one or more antibiotics are selected from any antibiotics that treat bacterial infections that express metallo beta-lactamase. In one embodiment, the one or more antibiotics are beta-lactam antibiotics. In one embodiment, the β-lactam antibiotic is selected from penicillin derivatives (penems), cephalosporins (cephems), monobactams, and carbapenems. In one embodiment, the β-lactam antibiotic is imipenem, ertapenem, meropenem, doripenem, biapenem, panipenem, ticarcillin, ampicillin, amoxicillin, carbenicillin, piperacillin, azurocillin, mezlocillin, ticofetacem, cefoperagem Selected.

  In another embodiment, the one or more antibiotics are carbapenem antibiotics. In one embodiment, the carbapenem antibiotic is selected from meropenem, biapenem, doripenem, panipenem, and imipenem.

相対立体化学を有する。 In one embodiment, the compound of formula I has the following:

Has relative stereochemistry.

In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are each H. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are independently C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC. (O) Selected from C _1-6 alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same and are C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC. (O) Selected from C _1-6 alkyl.

In one embodiment, when at least one of R ² , R ³ , R ⁴ , and R ⁵ is other than H, the compound of formula I is R ² , R ³ , R ⁴ , and R ⁵ are each H Is a prodrug of the active compound.

相対立体化学を有する。 In one embodiment, R ¹ is CH (NH ₂ ) C (O) OR ⁵ and R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C Selected from _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC (O) C _1-6 alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same. In one embodiment, when R ¹ is CH (NH ₂ ) C (O) OR ⁵ , the compound of formula I is:

Has relative stereochemistry.

In one embodiment, R ¹ is C (O) OR ⁵ and R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C _1-4 alkylene C _{5. -6} heterocycloalkyl, and _{C 1-4} is selected from alkylene -OC _{(O) C 1-6} alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same.

In one embodiment, R ¹ is C (O) NHR ⁵ and R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C _1-4 alkylene C _{5. -6} heterocycloalkyl, and _{C 1-4} is selected from alkylene -OC _{(O) C 1-6} alkyl. In one embodiment, R ² , R ³ , R ⁴ , and R ⁵ are the same.

である。 In one embodiment, heterocycloalkyl is

It is.

  In one embodiment, n, m, and p are each 1.

  In one embodiment, the compound of formula I is selected from AM-A, AM-B, and licomalasmin, or pharmaceutically acceptable salts and / or solvates thereof. In a further embodiment, the compound of formula I is a prodrug of AM-A, AM-B, or licomalasmin, or a pharmaceutically acceptable salt and / or solvate thereof.

  In one embodiment, one or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof, and one or more β Lactam antibiotics are formulated as separate pharmaceutical compositions for separate administration or use to a subject. In this embodiment, the separate pharmaceutical compositions are formulated independently of each other and according to the desired mode of administration, each effective. In one embodiment, the one or more beta-lactam antibiotics are formulated for administration or use by oral delivery or for delivery by infusion. In another embodiment, one or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof, are for administration or use by oral delivery or by infusion delivery. Formulated for.

  In one embodiment, one or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof, and one or more β The lactam antibiotic is formulated as a single pharmaceutical composition for combined simultaneous administration or use to a subject. In one embodiment, a single pharmaceutical composition is formulated for administration or use by oral delivery or infusion.

IV. Cell-Based Antibiotic Resistance Assay This application is also a cell-based screening assay comprising bacterial cells that express a bacterial resistance gene, wherein the cells encode (i) a protein that blocks molecular entry into the cells. And (ii) an assay that has been modified to delete one or more of a gene encoding a protein that facilitates export of the molecule from the cell.

In one embodiment, the bacterial cell is E. coli. E. coli cells. In a further embodiment, the gene encoding a protein that blocks entry of a molecule into the cell is bamB. In another embodiment, the gene encoding a protein that facilitates export of molecules from the cell is tolC. In a further embodiment, the bacterial resistance gene is a gene encoding a metallo β-lactamase, such as the bla _NDM-1 gene.

In one embodiment, the application is also a method of identifying a compound that treats antibiotic resistance comprising:
(A) contacting a bacterial cell expressing a bacterial resistance gene with one or more compounds, wherein the cell (i) a gene encoding a protein that blocks entry of the molecule into the cell and (ii) ) Modified so that one or more of the genes encoding the proteins that promote the export of the molecule from the cell are deleted, and the one or more compounds become proteins encoded by the bacterial resistance gene In contact with cells in the presence of sensitive antibiotics;
(B) identifying a compound that inhibits the growth of the bacterial cell as a compound for treating antibiotic resistance.

  In one embodiment, cells of bacterial cell growth by the compound are compared to a control.

  An antibiotic that is sensitive to a protein encoded by a bacterial resistance gene is an antibiotic whose potency is reduced in the presence of the protein encoded by the resistance gene.

  The following non-limiting examples are representative of the present application.

Examples Example 1: Isolation and activity of AM-A Materials and Methods (a) Reagents All enzyme and analytical grade chemicals were purchased from Sigma-Aldrich unless otherwise noted. Proton and carbon NMR spectra were recorded on a Bruker 700 MHz spectrometer. Nitrocefin was synthesized as previously reported.

(B) DNA manipulation and plasmid construction Plasmid DNA purification and gel extraction were performed using PureLink ™ Quick plasmid miniprep and PureLink ™ Quick gel extraction kit (Invitrogen), respectively. Restriction enzymes were purchased from Fermentas. PCR Primers for DNA amplification were purchased from IDT (Coralville, Iowa). PCR was performed using Phusion High Fidelity DNA Polymerase (Thermo Fisher Scientific) using the reaction conditions specified by the manufacturer. It was. All ligation reactions were performed using T4 DNA ligase (Thermo Fisher Scientific) according to the manufacturer's instructions. All β-lactamase overexpression constructs were generated without a leader peptide in the pET-28b plasmid containing an N-terminal His tag. The leader peptide sequence was determined using SignalP 4.0 25. All vectors were transformed into E. coli. E. coli TOP10 transformed into chemically competent cells.

(C) Protein purification VIM-2, IMP-7, CTX-M-15, TEM-1, and OXA-48: E. coli transformed with the respective β-lactamase construct. E. coli BL21 (DE3) colonies were inoculated with LB medium containing 50 μg / ml kanamycin and grown at 37 ° C. Protein expression was induced with 1 mM IPTG at OD600 0.7 and the cultures were incubated overnight at 16 ° C. Cells are harvested by centrifugation and the cell paste from a 1 L culture expressing β-lactamase is washed with 8 ml 0.85% NaCl, 20 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imidazole. And resuspended in buffer containing 20 μM ZnSO ₄ (for metalloenzyme) and then lysed by sonication. The lysate was centrifuged at 20,000 RPM (48254 × g) at 4 ° C. for 45 minutes using a Beckman JA 25.50 rotor. This supernatant was applied to a 5 mL HiTrap Ni-NTA column (GE Lifesciences) at a constant flow rate of 3 ml / min. The column was washed with 5 column volumes of the same buffer and an increasing imidazole step gradient was used for the washing and elution steps. Fractions containing purified β-lactamase are collected based on SDS-PAGE in a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 20% glycerol and 20 μM ZnSO ₄ for metalloenzymes. Dialyzed overnight at 4 ° C. NDM-1 was purified as described above with the addition of 84 μg of SUMO protease. Protease and uncleaved NDM-1 were removed by applying the dialyzed solution to a 5 ml HiTrap Ni-NTA column and collecting the effluent fraction. All purified enzymes were verified to be> 95% pure as assessed by SDS-PAGE and stored at -20 ° C.

(D) Cell-based screening ~ 500 natural product extracts in combination with 0.125 μg / ml meropenem. E. coli BW25113ΔbamBΔtolCΔaraDAB :: pLac (blaNDM-1). This screening was performed in duplicate in 96 well plates using cation-controlled Mueller Hinton broth (CAMHB). The low growth control was 2 × MIC meropenem (1 μg / ml) and the growth control was 1/4 MIC meropenem (0.125 μg / ml). Z ′ has been determined to be 0.77, which showed a good screening window.

旋光度は、パーキンエルマー ２４１旋光計で決定した。質量を、Ｍａｘｉｓ ４Ｇ Ｑ／ＴＯＦ、陽イオンモードでのＥＳＩ ＭＳ直接注入（３μＬ／分）を使用して決定した。 (E) Purification of AM-A WAC-138 (Aspergillus versicolol) (4 L) was evaporated under reduced pressure with 2% (W / V) HP-20 resin (Diaion) to obtain residual WAC138-E. It was. The crude mixture was eluted with H ₂ O (1 L), 10% MeOH (1 L), 25% MeOH (1 L), 60% MeOH (1 L), and 100% MeOH (1 L) HP-20 ( 100 g) applied on the column to obtain 5 fractions, WAC138-E-1-5. Active fraction WAC138-E-1 was applied to reverse phase CombiFlash ISCO (RediSep Rf C18, Teledyne) and eluted with a water-acetonitrile linear gradient system (0-100% acetonitrile) with 65 Fractions, WAC138-E-1-1-165 were obtained. The active subfraction WAC138-E-1-5 was passed through a Sephadex LH-20 column (100 ml) eluting with 25% MeOH to obtain 12 subfractions. Active subfractions WAC138-E-1-5-6-8 were combined and recrystallized in 5 ml of 1% acetic acid (V / V) to give AM-A as white crystals. From 1 L of culture, ˜200 mg of AM-A was obtained.

The optical rotation was determined with a Perkin Elmer 241 polarimeter. Mass was determined using Maxis 4G Q / TOF, ESI MS direct injection (3 μL / min) in positive ion mode.

(F) IC ₅₀ enzyme inhibition assay Enzyme (NDM-1, 5 nM; VIM-2, 500 pM; CTX-M-15, 500 pM; KPC-2, 5 nM; OXA-48, 1 nM; TEM-1, 100 pM; ACE, 50 nM) after preincubation with AM-A for 5-10 minutes, followed by 30 μM nitrocefin (100 μM nitrocefin for TEM1, 250 μM furanacryloyl-L-phenylalanylglycylglycine [FACPG] for ACE24) ). The metalloenzyme was supplemented with 10 μM ZnSO ₄ . The assay was read in the form of a 96-well microplate at 490 nm using a Spectramax reader (Molecular Device) at 30-37 ° C.

(G) Incubation of metalloenzyme with AM-A The enzyme (500 nM) was incubated with AM-A (500 μM) for 10 minutes. 20 μl of the above was diluted with 180 μl of nitrocefin or FPGG substrate to give the following final concentrations: enzyme (50 nM), FPGG (50 μM) / nitrocefin (20 μM), AM-A (50 μM). Buffer (50 mM HEPES pH 7.5, 300 mM NaCl) was stirred overnight with 2 g / 100 mL of Kilex-100 (Bio-Rad; Richmond, CA). The assay was read in the form of a 96-well microplate at 490 nm using a Spectramax reader (Molecular Device) at 37 ° C.

(H) Reversibility assay 5 mL of NDM-1 (500 nM) was incubated for 1 hour on ice with or without AM-A (100 μM). The control without enzyme was buffer alone (Chirex treated 50 mM HEPES pH 7.5). After equilibrating the column with buffer, 2.5 ml was passed through a PD-10 spin column (GE Healthcare) and centrifuged at 2,000 × g for 2 minutes. Before PD-10 (+ AM-A, -AM-A, -NDM-1), after PD-10 ((+ AM-A, -AM-A, -NDM-1), and nitrocefin were equilibrated to 30 ° C. 20 μl of the enzyme solution was added to 180 μl of nitrocefin to give a final enzyme concentration of 50 mM and 100 μM nitrocefin and 10 μM AM-A The assay was performed at 30 ° C. using a Spectramax reader (Molecular device). Read in 96-well microplate format at 490 nm for 1 hour.

(I) Zn ²⁺ recovery assay NDM-1 (5 nM) supplemented with 10 μM ZnSO ₄ was incubated with 20 μM AM-A at 30 ° C. for 15 minutes. Nitrocefin (30 μM) and 500 nM to 40 μM ZnSO ₄ were added to a final volume of 100 μl, and the absorbance at 490 nm was monitored using a Spectramax reader (Molecular Device) at 30 ° C. for 30 minutes. % Residual activity was calculated from the control without AM-A. Slightly negative% residual activity was reported as 0.

(J) Inactivation kinetics NDM-1 (50 nM) was added to 20 μM nitrocefin containing AM-A at a serial dilution of 8 μM to 1/2. The assay was performed in 50 mM HEPES pH 7.5, 200 μL final volume. The assay was read in the form of a 96-well microplate at 490 nm for 10 minutes at 37 ° C. using a Spectramax reader (Molecular device). VIM-2 (10 nM) was added to 20 μM nitrocefin containing AM-A at a serial dilution of 1/2 from 16 μM. The assay was performed in 50 mM HEPES (pH 7.5), 200 μL final volume. The assay was read in the form of a 96 well microplate at 490 nm for 10 minutes at 37 ° C. using a Spectramax reader (Molecular Device) as previously reported. In all assay experiments, the offset between the start of the reaction and the first reading was ~ 6s.

であって、式中、
Ｅ・Ｚｎ、Ａ、Ｅ・Ｚｎ・Ａ、およびＺｎ・Ａは、それぞれ、活性金属酵素、ＡＭ‐Ａ、三元の金属酵素‐ＡＭ‐Ａ複合体、およびＡＭ‐Ａ−金属複合体である、
モデルにしたがい、ＡＭ‐Ａ濃度に関する擬一次速度定数ｋｉの依存性に基づき計算した。Ｋｉは三元複合体の解離定数を表し、ｋ_＋２は、三元複合体の不活化酵素（Ｅ_{ｉｎａｃｔ}）およびＡＭ‐Ａ−Ｚｎ複合体への解離の速度定数である。定常状態の進行曲線は、以下の積分方程式

であって、式中、ｖ_０はレポーター基質の代謝回転の初期速度であり、ｋ_ｉは偽一次不活性化速度定数である、
式に適合した。Ｋ_ｉおよびｋ_＋２の個々の値は、以前に報告されたように^２５式２

であって、式中、［Ａ］はＡＭ‐Ａの濃度であり、［Ｓ］およびＫ_Ｍはそれぞれレポーター基質の濃度およびＫ_Ｍである、
式２にｋ_ｉの値を適合することにより決定した。 Rate constants that characterize enzyme inactivation can be expressed as

And in the formula,
E · Zn, A, E · Zn · A, and Zn · A are active metalloenzyme, AM-A, ternary metalloenzyme-AM-A complex, and AM-A-metal complex, respectively. ,
According to the model, the calculation was based on the dependence of the pseudo first-order rate constant ki on the AM-A concentration. Ki represents the dissociation constant of the ternary complex, and k ₊₂ is the rate constant of dissociation of the ternary complex into the inactivated enzyme (E _inact ) and the AM-A-Zn complex. The steady-state progression curve is the integral equation

Where v ₀ is the initial rate of reporter substrate turnover and k _i is the pseudo first order inactivation rate constant,
Fit the formula. The individual values for K _i and k ₊₂ are ²⁵ equations 2 as reported previously.

A is wherein, [A] is a concentration of AM-A, a [S] and _{K M} concentration of each reporter substrate and _{K M,}
It was determined by fitting the values of k _i in Equation 2.

(K) ICP Mass Spectrometry Inductively coupled mass spectrometry (ICP-MS) was used to analyze the nature of AM-A chelating Zn ⁶⁶ from purified NDM-1 (27-270). NDM-1 protein was purified ²⁶ as previously reported, and freshly added to ICP-MS buffer (20 mM HEPES, 100 mM NaCl, pH 7.5) using a 15 kDa cut-off dialysis tube. Exchanged overnight at 0 ° C. to remove contaminating metal. The protein was concentrated to ˜5 mg / ml and various concentrations of AM-A were incubated with protein samples in triplicate for 3 hours at room temperature with gentle agitation. A sample of protein-AM-A was dialyzed again overnight at 4 ° C. into ICP-MS buffer using a 12-14 kDa cutoff D-tube dialyzer mini (EMD biosciences) microdialysis cassette. The final protein was diluted to 1 mg / ml in ICP-MS buffer and then diluted 1/40 in internal standard (10 ug / L Sc45, 1% nitric acid, Inorganic Ventures). Prior to analysis of the samples, the ICP MS was calibrated using a standard solution (Inorganic Ventures) containing the isotopes of the metals of interest. The protein sample was then transferred by spray to a NexION 3000 ICP mass spectrometer (Perkin Elmer). Quantitative analysis was performed in triplicate for each sample at 60 sweeps per reading using a peak hopping mode with an acquisition time of 50 ms / AMU for each element. The instrument settings were rf power (1600 W), integration time (35 s), collision gas (Ar40), RPQ voltage (25 V) and sample flow rate (4 rpm). Isotope abundances were determined by integrating peak areas using the NexION software program and the data presented as a chart using Microsoft Excel.

(L) Determination of FIC index FIC values were determined by standard method ²⁷ of setting up a checkerboard with 8 concentrations of each meropenem and AM-A serially diluted by 1/2. The experiment was performed in duplicate and the average value was used for the calculation. The MIC for each compound was the lowest compound concentration that did not show growth. The FIC for each compound was calculated as the compound concentration in the presence of the co-compound for wells showing no growth and divided by the MIC for that compound. The FIC index is the sum of two FICs.

(M) at various concentrations of screening AM-A clinical isolates, were selected in combination with Meropenem of a limit point of EUCAST for resistance 2 mg / l ^28. The synergistic properties of the two compounds were tested in microtiter trays using BHI (Oxford Science Park, UK) and 0.5 McFarland inoculum as growth media. All plates contained the control strains E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Overall, 226 non-clonal clinical isolates (Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter) were obtained from the following MBL: SPM-1 (n = 17), AIM-1 (n = 8), One of NDM-1 (n = 67), VIM-type (n = 114) or IMP-type (n = 20) was tested. Three E.V. E. coli and 5 Klebsiella pneumoniae also have serine carbapenemase KPC and 4 E. coli harboring NDM-1. Coli and one K. pneumoniae also had carbapenemase OXA-181. Plates were incubated at 37 ° C. and read after 18 hours. Subset strains (# 48) were repeated to test reproducibility and showed no deviation from the original data.

(N) Animal Experiments All animals were housed in specific pathogen-free units at the Central Animal Facility at MacMaster University. All experimental protocols were approved by the McMaster Animal Research Ethics Board and performed accordingly. Female CD1 mice were purchased from Charles River.

(O) Bacterial infection Mice were infected with Klebsiella pneumoniae N11-2218 at a dose of 2 × 10 ⁶ colony forming units (cfu) in all organ bacterial challenge experiments, or 5 × 10 ⁷ cfu in all survival experiments. Infection via the abdominal cavity (ip). In all organ bacterial challenge experiments, mice were euthanized 48 hours after infection and spleen and liver were collected. Organs were placed on ice in 1 ml sterile PBS and then homogenized (Mixer Mill 400; Lecce). Organ homogenates were serially diluted with PBS and plated on brilliant green agar (Oxoid) for cfu measurement. Mice were monitored to endpoint for survival curves. In all experiments, mice were treated 30 minutes after infection with either specific subcutaneous doses of PBS, meropenem, AM-A inhibitors, or a combination of antibiotics and inhibitors.

Results and discussion E. coli strain BW25113 was modified by deletion of the bamB and tolC genes to increase permeability and reduce small molecule excretion. This strain was then further modified by single copy chromosomal insertion of the bla _NDM-1 gene under the control of the pLac promoter. Microbial natural product extracts were screened against this strain in the presence of sublethal ¼ MIC meropenem. AM-A was purified from the hit extract and its structure was revealed by NMR, mass spectrometry and optical rotation analysis. The IC ₅₀ value of AM-A was determined using the following purified enzymes: MBL IMP-7, VIM-2, and NDM-1; SBL CTX-M-15, KPC-2, OXA-48, and TEM-1, and For ACE from rabbit lungs, determinations were made using nitrocefin (for MBL and Ser BL) and furanacryloyl-L-phenylalanylglycylglycine (for ACE) ²¹ as reporter substrates. IC ₅₀ , reversibility, Zn ²⁺ recovery, and inactivation enzyme assays were performed in 50 mM HEPES (pH 7.5) and measured using a SpectraMax reader (Molecular Device). ICP-MS experiments were performed using 5 mg / ml purified NDM-1 and various concentrations of AM-A, followed by transfer into a NexION 3000 ICP mass spectrometer (Perkin Elmer) by dilution and spraying. It was. FIC values were determined using standard method ²⁷ . Various concentrations of AM-A were tested in combination with 2 mg / l meropenem against 200+ MBL-expressing clinical isolates including Pseudomonas, Acinetobacter, and Enterobacteriaceae. A 2 × 10 ⁶ forming unit (cfu) dose of Klebsiella pneumoniae N11-2218 was used in all organ bacterial load experiments and a dose of 5 × 10 ⁷ cfu was used in all survival experiments. In all experiments, mice were treated with compounds 30 minutes after infection.

AM-A showed potent dose-dependent inhibition of NDM-1 and related MBL VIM-2 (FIG. 1b) in vitro, indicating weaker activity against IMP-7 MBL. AM-A was ineffective against serine β-lactamases TEM-1 and CTX-M-15, and serine carbapenemases KPC-2 and OXA-48 (FIG. 1b). Inhibition of NDM-1 was shown to be irreversible after removal of AM-A by gel filtration (FIG. 1c), but enzyme activity was restored by the addition of excess ZnSO ₄ consistent with the metal depletion mechanism It was possible (FIG. 1d). Although inhibition of mammalian metalloenzymes can be observed as a potential side effect, AM-A was only able to reduce the activity of ACE in rabbit lungs by ˜35% in a concentration response assay. Incubation of metalloenzymes NDM-1, VIM-2, IMP-7, and ACE with extended concentrations of AM-A (0.5 mM) in ²⁹ Zn ²⁺ deficient buffer prepared as previously reported Results in complete inactivation of NDM-1 and VIM-2 and inhibition of ˜70% and ˜50% of the activity of IMP-7 and ACE, respectively, with selectivity for NDM and VIM MBL. Illustrated. Time-dependent inactivation is saturating with respect to NDM-1 (K _i = 11 nM, k ₊₂ = 0.0062 s ⁻¹ ) and VIM-2 (K _i = 7 nM, k ₊₂ = 0.0065 s ⁻¹ ). It was shown that AM-A was consistent with an inactivation mechanism that removes Zn ²⁺ . This mechanism of action was confirmed by inductively coupled mass spectrometry which showed a loss of ˜1.8 Zn equivalent in NDM-1 inactivated by AM-A.

E. genetically engineered E. coli A systematic titration of AM-A and meropenem concentrations against E. coli and clinical CRE strains showed that AM-A restored meropenem activity consistent with NDM inhibition. The checkboard MIC test showed the expected synergy between meropenem and AM-A only with CRE expressing NDM-1 and not with carbapenem sensitive strains (FIGS. 2a, b). Fractional inhibitory concentration (FIC) index values were determined to be <0.1 in 16 clinical CRE isolates tested against the combination of meropenem and AM-A (≦ 0.5) FIC values are defined as synergistic ²⁷ ). Synergism of AM-A (8 μg / ml) with meropenem, 229 MBL positive (SPM-1, IMP, NDM, AIM and VIM) non-clonal clinical isolates (Enterobacteriaceae, Acinetobacter), and Further investigation was made using Pseudomonas) (FIG. 2c). Seventy-six isolates with serine carbamase or MBL and serine carbapenemase were also tested. Strains were collected over 10 years as part of a global MBL collection including isolates from Russia, India, Pakistan, Australia, North Africa, and South America. AM-A restored meromenem sensitivity (2 μg / ml) in 88% of NDM positive isolates and 90% of VIM positive isolates. Importantly, AM-A was active in Pseudomonas (primarily Pseudomonas aeruginosa), which is considered a very difficult model for new antibiotics. AM-A showed little synergy with SPM-1, IMP and AIM, but these MBLs were less than “worldwide” VIM and NDM MBL, and therefore not clinically relevant It is regarded. Lack of synergy with IMP expressing strains correlates well with biochemical data indicating weak inactivation of purified IMP-8 compared to NDM-1 or VIM-2.

  Because of the resistance profile of NDM-1 positive clinical CRE and the potency that AM-A enhances meropenem activity against NDM-1 positive clinical CRE, AM-A exhibits NDM-1-mediated resistance to meropenem in vivo. It is suggested to reverse and restore the clinical efficacy of this antibiotic. To test this, CD1 mice were infected intraperitoneally with a lethal dose of NDM-1 positive Neisseria pneumoniae N11-2218, causing a fatal systemic infection, meropenem or AM-A monotherapy, or antibiotics The efficacy of the agent-inhibitor combination therapy was evaluated. From preliminary dose experiments, the bacterial load of NDM-1-positive Klebsiella pneumoniae in tissues is not affected by treatment with AM-A alone, and this strain is a meropenem monotherapy at doses below 50 mg / kg. It has been empirically determined to be resistant to and cause fatal infections. However, combination therapy including AM-A and meropenem significantly reduced bacterial load in the spleen after a single intraperitoneal administration (FIG. 3a) and to a lesser extent in the liver (FIG. 3b). ). Of note, meropenem or AM-A alone did not prevent lethal infections caused by NDM-1-positive Klebsiella pneumoniae, but single-dose combination therapy resulted in> 95% survival on day 5 post infection. (Fig. 3c).

AM-A is a non-toxic candidate for antibiotic adjuvants that can overcome resistance mediated by NDM and VIM MBL and resensitize carbapenem-resistant gram-negative pathogens to carbapenem. Active drug / inhibitor combinations continue to be very successful in the clinic with inhibitors targeted to Ser-beta lactamase ³⁰ . AM-A is the first to show in vitro and in vivo complementary activity against important MBL that has spread rapidly worldwide and has resulted in significant human morbidity, especially in developing countries. When used in combination with β-lactam antibiotics such as meropenem as shown herein, resistance could be overcome and antibiotic activity was fully restored. AM-A, or a semi-synthetic derivative, is therefore an excellent clue to antibiotic adjuvant cotherapy to address the recent emergence of MBL in the clinic.

であって、式中、
Ｒ＝−ＣＨ_３、−（ＣＨ_２）_ｘＣＨ_３（ｘ＝１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１５、１５、１６、１７または１８）、−ＣＨ_２ＯＣ（Ｏ）ｔ−Ｂおよび

である、
ＡＭ‐Ａのプロドラッグを得る。 Example 2 Prodrugs of AM-A In this example, various prodrugs of AM-A are prepared.
(A) General chemistry A prodrug of AM-A is prepared by activating the carboxylic acid group in AM-A and then adding the appropriate nucleophile in the presence of a base. The reaction mixture is isolated after neutralization and the following prodrugs of AM-A

And in the formula,
R = —CH ₃ , — (CH ₂ ) _x CH ₃ (x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 15, 16, 17 or _{18), - CH 2 OC (} O) t-B and

Is,
A prodrug of AM-A is obtained.

  Although the present application has been described with reference to examples, the claims are not to be limited by the embodiments described in the examples, but have the broadest interpretation consistent with the description as a whole. It should be understood that it should.

  All publications, patents, and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was intended to be incorporated by reference in its entirety. Is done. If it is found that the terms of the present application are defined differently from the document incorporated by reference herein, the definitions provided herein shall be treated as the definitions of the terms. .

Citation of documents related to this specification

Claims (34)

One or more beta-lactam antibiotics;
One or more formulas I :

A compound of the formula:
R ¹ is _{C H (NH 2) C (} O) OR 5;
R ² , R ³ , R ⁴ and R ⁵ are independently H, C _1-24 alkyl, C _1-6 alkylene C _6-10 aryl, C _1-6 alkylene C _3-10 cycloalkyl, C _{1 -6} alkylene _{C 3-10} heterocycloalkyl, and _{C 1-6} Ru is selected from alkylene -OC _{(O) C 1-6} alkyl,
A compound, or a pharmaceutically acceptable salt and / or solvate thereof ,
A pharmaceutical composition for treating a bacterial infection or a disease, disorder or condition resulting from a bacterial infection .   The pharmaceutical composition according to claim 1, wherein the one or more β-lactam antibiotics are selected from penicillin derivatives (penems), cephalosporins (cephems), monobactams, and carbapenems.   The one or more beta-lactam antibiotics are imipenem, ertapenem, meropenem, doripenem, biapenem, panipenem, ticarcillin, ampicillin, amoxicillin, carbenicillin, piperacillin, azurocillin, mezlocillin, ticofuracin, cefotaxon, 3. A pharmaceutical composition according to claim 2 selected from ceftazidime.   The pharmaceutical composition according to claim 2, wherein the one or more antibiotics are carbapenem antibiotics.   The pharmaceutical composition according to claim 4, wherein the carbapenem antibiotic is selected from meropenem, biapenem, doripenem, panipenem, and imipenem. The pharmaceutical composition according to any one of claims 1 to 5 , wherein R ² , R ³ , R ⁴ , and R ⁵ are each H. R ² , R ³ , R ⁴ , and R ⁵ are independently C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC (O) C _1. The pharmaceutical composition according to any one of claims 1 to 5 , which is selected from _-6 alkyl. R ² , R ³ , R ⁴ , and R ⁵ are the same, and C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC (O) C _1- 8. A pharmaceutical composition according to claim 7 selected from ₆ alkyls. R ² , R ³ , R ⁴ , and R ⁵ are independently H, C _1-18 alkyl, C _1-4 alkylene C _5-6 heterocycloalkyl, and C _1-4 alkylene-OC (O). _6. The pharmaceutical composition according to any one of claims 1 to 5 , which is selected from C1-6 alkyl. ^{R 2, R 3, R 4} , and ^{R 5} are the same, pharmaceutical composition according to claim 9. Compounds of formula I, AM- A or is selected from those pharmaceutically acceptable salts and / or solvates, pharmaceutical composition according to any one of claims 1-5. One or more beta-lactam antibiotics;
And one or more compounds of formula I or their pharmaceutically acceptable salts and / or solvates thereof, according to any one of claims 1 and claims 6 to 11,
A pharmaceutical composition comprising
Said one or more β-lactam antibiotics and one or more compounds of formula I are effective in the treatment of a bacterial infection or a disease, disorder or condition resulting from a bacterial infection. Present in an effective amount to improve,
Pharmaceutical composition. 13. The pharmaceutical composition according to any one of claims 1 to 12 , wherein the one or more beta-lactam antibiotics and the one or more compounds of formula I are formulated in separate dosage forms. . 13. The pharmaceutical composition according to any one of claims 1 to 12 , wherein the one or more beta-lactam antibiotics and the one or more compounds of formula I are formulated in a single dosage form. object. 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the bacterial infection is an infection of a bacterium that expresses at least one metallo β-lactamase (MBL). 16. The pharmaceutical according to claim 15 , wherein the MBL is IMP type, Verona integron-coded metallo β-lactamase (VIM), or New Delhi metallobeta lactamase (NDM). Composition . 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the bacterial infection is an infection of at least one carbapenem resistant gram negative bacterium. The pharmaceutical composition according to any one of claims 1 to 14, wherein the bacterial infection is an infection of at least one bacterium belonging to the family Enterobacteriaceae, Acinetobacter, or Pseudomonas. 19. The pharmaceutical composition according to claim 18 , wherein the Enterobacteriaceae bacterium is Klebsiella. 19. The pharmaceutical composition according to claim 18 , wherein the enterobacteriaceae bacterium is Klebsiella pneumonia or Escherichia coli . The pharmaceutical composition according to claim 18 , wherein the Pseudomonas bacterium is Pseudomonas aeruginosa. A pharmaceutical composition for use in a method of treating or preventing a disease, disorder or condition resulting from a bacterial infection in a subject comprising:
12. The composition comprises one or more compounds of formula I according to claim 1 and any one of claims 6-11, or pharmaceutically acceptable salts and / or solvates thereof. ,
The method comprises administering to the subject an effective amount of one or more beta-lactam antibiotics in combination with the composition ;
Pharmaceutical composition . A pharmaceutical composition for use in a method for improving the efficacy of a β-lactam antibiotic for treating a bacterial infection comprising:
The composition comprises one or more compounds of formula I or a pharmaceutically acceptable salt and / or solvate, as defined in any one of claims 1 and claims 6 to 11 ,
The method includes administering the composition in combination with the antibiotic to a subject in need thereof.
Pharmaceutical composition . A pharmaceutical composition for use in a method of improving the efficacy of a β-lactam antibiotic for treating a disease, disorder or condition resulting from a bacterial infection comprising:
The composition comprises one or more compounds of formula I or a pharmaceutically acceptable salt and / or solvate, as defined in any one of claims 1 and claims 6 to 11 ,
The method includes administering the composition to a subject in need thereof .
Pharmaceutical composition . A kit for treating a bacterial infection or a disease, disorder or condition resulting from a bacterial infection ,
One or more beta-lactam antibiotics;
The compound of claim 1 and one or more of the formula I as defined in any one of claims 6-11 or a pharmaceutically acceptable salt and / or solvate thereof,
Optionally, the one or more beta-lactam antibiotics and the one or more compounds of formula I, or pharmaceutically acceptable salts and / or solvates thereof, to a subject in need thereof. A kit comprising instructions for administration. A kit for treating a bacterial infection or a disease, disorder or condition resulting from a bacterial infection ,
The compound of claim 1 and one or more of the formula I as defined in any one of claims 6-11 or a pharmaceutically acceptable salt and / or solvate thereof,
Administering said one or more compounds of formula I, or a pharmaceutically acceptable salt and / or solvate thereof, a bacterial infection or an antibiotic for a disease, disorder or condition resulting from a bacterial infection And an instruction for administration to the subject. A kit for improving the efficacy of a β-lactam antibiotic for treating a bacterial infection, or a disease, disorder or condition resulting from a bacterial infection ,
One or more beta-lactam antibiotics;
The compound of claim 1 and one or more of the formula I as defined in any one of claims 6-11 or a pharmaceutically acceptable salt and / or solvate thereof,
Optionally, the one or more beta-lactam antibiotics and the one or more compounds of formula I, or pharmaceutically acceptable salts and / or solvates thereof, to a subject in need thereof. A kit comprising instructions for administration. A kit for improving the efficacy of a beta-lactam antibiotic for the treatment of a bacterial infection or a disease, disorder or condition resulting from a bacterial infection ,
The compound of claim 1 and one or more of the formula I as defined in any one of claims 6-11 or a pharmaceutically acceptable salt and / or solvate thereof,
One or more compounds of formula I as defined above, or pharmaceutically acceptable salts and / or solvates thereof, to treat a bacterial infection or a disease, disorder or condition resulting from a bacterial infection And a manual for administration to a subject being administered the β-lactam antibiotic. 29. Kit according to any one of claims 25 to 28 , wherein the one or more beta-lactam antibiotics are selected from penicillin derivatives (penems), cephalosporins (cephems), monobactams and carbapenems. . One or more beta-lactam antibiotics are imipenem, ertapenem, meropenem, doripenem, biapenem, panipenem, ticarcillin, ampicillin, amoxicillin, carbenicillin, piperacillin, azurocillin, mezlocillin, tifocilimone, cefodizem The kit according to any one of claims 25 to 28 , which is selected. 31. A kit according to any one of claims 25 to 30 , wherein the one or more beta-lactam antibiotics and the one or more compounds of formula I are formulated in separate dosage forms. 31. A kit according to any one of claims 25 to 30 , wherein the one or more beta-lactam antibiotics and the one or more compounds of formula I are formulated in a single dosage form.   25. A pharmaceutical composition according to any one of claims 1 to 24, wherein the compound of formula I is a natural product extracted from a fungus.   34. A pharmaceutical composition according to claim 33, wherein the fungus is Aspergillus versicolor.

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